Project summary This proposal represents an opportunity to directly study in vivo how the intermediary metabolite nicotinamide adenine dinucleotide (NAD+) contributes to the onset of age-related pathologies, due to its limited local availability. NAD+ is critical for cellular metabolism and for prolonging health in old age. Decreased steady- state NAD+ levels have been linked to human pathologies such as neurodegeneration, cardiovascular disease, metabolic syndrome, and cancer. However, NAD+ concentrations are highly compartmentalized by cell type, subcellular localization, and protein-bound or free fractions. Thus, a lack of methods to directly monitor free NAD+ in cells with spatial and temporal information has hindered our learning about the relevant pools, threshold concentrations, and timing that NAD+ may undergo leading to disease onset. This precludes our ability to identify treatments or approaches to intervene before NAD+ levels are misregulated. To address this roadblock, we have developed a genetically-encodable fluorescent biosensor for free NAD+. Targeting of the sensor to subcellular compartments has revealed compartmentalization of intracellular NAD+ that is differentially coordinated in different cell types. Excitingly, the data also pointed to the existence of a putative mammalian mitochondrial NAD+ transporter. We believe the sensor can address key problems in aging with the hypothesis that misregulation of mitochondrial NAD+ is a major underlying cause of late-onset pathologies. This proposal will use the sensor to identify the proteins required for mitochondria to sustain their elevated concentrations of NAD+ and how this pool may be altered in older neurons. This has important implications for understanding cellular bioenergetics and mitochondrial health, as well as identifying new options to combat neurodegeneration. Understanding how local NAD+ levels fluctuate and may limit the function of NAD+-dependent enzymes is important to evaluate whether NAD+ regulation represents a viable treatment approach or intervention for age-related diseases.